Selection markers and selection systems are widely used in genetic engineering, recombinant DNA technology and production of recombinant products, for example antibodies, hormones and nucleic acids, in eukaryotic cell culture. The primary goal of such dominant selection markers and selection systems is to introduce a selectable gene which upon exposure to selective growth conditions provides cells capable of high-level production of the recombinant products of interest.
To date, there are 3 major selection marker systems available:
(a) The glutamine synthetase system: The enzyme glutamine synthetase (GS) is responsible for the biosynthesis of glutamine from glutamate and ammonia. This biosynthetic reaction provides the sole pathway for glutamine formation in mammalian cells. Thus, in the absence of glutamine in the growth medium, the enzyme GS is essential for the survival of mammalian cells in culture. Importantly, certain mammalian cell lines including mouse myeloma cells lack the expression of sufficient GS and thus cannot survive without exogenously added glutamine. Hence, such a cell line is an suitable acceptor for a transfected GS gene that in this system can function as a selectable marker that allows for cell growth in a medium lacking glutamine. In contrast, cell lines such as the widely used Chinese hamster ovary (CHO) cells express sufficient GS to support growth in glutamine-free medium. Therefore, if these CHO cells are to be used as the recipient cells for the transfection of the GS gene, the specific and potent GS inhibitor methionine sulfoximine (MSX) can be applied in order to inhibit endogenous GS activity such that only transfectants expressing high levels of the transfected GS gene can survive in a glutamine-free medium. A major disadvantage of the GS system is the relatively long time (i.e. 2-6 months) of selective growth in order to establish cells stably overexpressing the target gene of interest. Another disadvantage is the frequent utilization of the cytotoxic agent MSX for the augmentation of the selective pressure. The presence of such a cytotoxic agent along with a recombinant product of interest (e.g. a polypeptide like an antibody) may require additional purification steps to rid of this cytotoxic agent.
(b) The dihydrofolate reductase/MTX selection system: Dihydrofolate reductase (DHFR) catalyzes the NADP-dependent reduction of dihydrofolic acid to tetrahydrofolic acid (THF). THF is then interconverted to 10-formyl-THF and 5,10-methylene-THF which are used in the de novo biosynthesis of purines and thymidylate, respectively. DHF is the byproduct of the catalytic activity of thymidylate synthase (TS) which catalyzes the conversion of dUMP to dTMP in a 5,10-methylene-THF-dependent reaction. Thus, DHFR is crucial for the recycling of THF cofactors that are essential for the biosynthesis of purine and pyrimidine nucleotides that are necessary for DNA replication. Hence, cells (e.g. CHO cells) that lack the DHFR gene (i.e. by targeted genomic deletion) can be used as recipients for the transfection of the DHFR gene in a medium that is free of nucleotides. After transfection, the cells can be subjected to a gradual increase in the concentrations of the antifolate MTX, a most potent DHFR inhibitor (Kd=1 pM), thereby forcing the cells to produce increased levels of DHFR. Upon multiple rounds of selection, the selectable marker DHFR frequently undergoes significant gene amplification. Furthermore, a mutant mouse DHFR with a major resistance to MTX has also been extensively used as a dominant selectable marker that markedly enhances the acquisition of high level MTX-resistance in transfectant cells. A major disadvantage of the DHFR/MTX selection system is that this technique utilizes a mutagenic cytotoxic agent, MTX, that can readily alter the genotype of the recipient cells. Additionally, specific safety measures may have to be taken to protect the persons handling such agents. This frequently results in MTX-resistant cell populations in which no expression of the target gene of interest is present due to loss of function mutations in the reduced folate carrier (RFC) and/or loss of RFC gene expression, both of which abolish MTX uptake. Another disadvantage is that the mutagenic drug MTX may readily contaminate the secreted overexpressed target product (e.g. a polypeptide like an antibody) contained in the growth medium thereby requiring labor intensive, time-consuming and expensive chromatographic methods necessary to rid off this mutagenic compound, MTX. In addition, the absence of MTX in the final product has to be demonstrated by respective assays.
(c) The reduced folate carrier selection system: The reduced folate carrier (RFC) is a ubiquitously expressed membrane glycoprotein that serves as the major transporter for the uptake of reduced folates such as 5-methyl-THF and 5-formyl-THF. However, RFC displays a very poor affinity for the oxidized folate, folic acid. Hence, cells that lack the expression of RFC or have been deleted for the genomic RFC locus can serve as recipients for the transfection of the selectable marker gene RFC under conditions in which reduced folates such 5-formyl-THF are gradually deprived from the growth medium thereby forcing the cells to express increased levels of the this folate transporter. There are several disadvantages for the RFC selection system: a) One must use RFC-null recipient cells in which the endogenous RFC locus has been knocked out or inactivated by targeted knockout or loss of function mutations. b) RFC has an extremely poor transport affinity for folic acid and thus this oxidized folate cannot be used for selection. c) As opposed to the current folate-receptor based system that is a unidirectional folate uptake system and which will be explained in detail below, RFC is a bi-directional folate transporter that exhibits equally potent import and export of folates. This implies that under conditions of folate deprivation, RFC overexpression may be detrimental to the recipient cells that further export folate via the overexpressed RFC.
The aim of the present invention is to provide a novel metabolic selection system that has certain advantages over the prior art selection systems mentioned above. The novel selection system is based upon the use of folates in the cell culture medium and on the presence of folate receptors introduced via an expression vector into the recombinant eukaryotic cell intended to produce a product of interest. This novel approach requires no prior deletion of an endogenous folate receptor (FR) gene. Following the introduction of a vector harboring both the FR selectable gene as well as the polynucleotide encoding a product of interest (like a polypeptide), cells are grown in a selective medium containing highly limiting concentrations of folates. Hence, only cells that markedly overexpress FR can take up sufficient folates to sustain cell growth, DNA replication and cellular proliferation, thereby allowing for overexpression of the target product of interest.
The oxidized folate, i.e. folic acid, as well as reduced derivatives of folic acid, known as reduced folates or tetrahydrofolates (THF) are a group of B-9 vitamins that are essential cofactors and/or coenzymes for the biosynthesis of purines, thymidylate and certain amino acids in eukaryotic, in particular mammalian, cells. THF cofactors are particularly crucial for DNA replication and hence cellular proliferation. Specifically, THF cofactors function as donors of one-carbon units in a series of interconnected metabolic pathways involving de novo biosynthesis of purines and thymidylate, amino acids as well as methyl group metabolism, including CpG island methylation of DNA. Specifically, THF cofactors including 10-formyl-THF (10-CHO-THF) contribute one-carbon units in two key de novo formyltransferase reactions involved in the de novo biosynthesis of purines. The first enzyme, glycinamide ribonucleotide transformylase (GARTF), is involved in the formation of the imidazole ring of purines, whereas the more downstream reaction mediated by 5-aminoimidazole-4-carboxamide ribonucleotide transformylase (AICARTF) yields the purine intermediate inosine 5′-monophosphate (IMP). The latter serves as a key precursor for the regulated biosynthesis of AMP and GMP. Furthermore, 5,10-methylene-THF (5,10-CH2-THF), is another important THF coenzyme which functions as a crucial cofactor for the enzyme thymidylate synthase (TS). TS catalyzes the formation of thymidine monophosphate (dTMP) from dUMP. Hence, these folate-dependent enzymes are key mediators of the de novo biosynthesis of purine and thymine nucleotides essential for DNA replication. As such, these folate-dependent enzymes were identified as targets for the activity of folic acid antagonists known as antifolates. For example, the 4-amino folic acid analogue aminopterin and its homologue 4-amino-10-methylfolic acid, methotrexate (MTX) were the first class of antimetabolites that were introduced to the clinic for the chemotherapeutic treatment of childhood acute lymphoblastic leukemia (ALL). Antifolates are currently key components of different chemotherapeutic regimens currently used for the treatment of other human malignancies including osteosarcoma, breast cancer, primary central nervous system lymphoma, choriocarcinoma and gestational trophoblastic neoplasia.
In contrast to most prokaryotes, plants, fungi and certain protests which synthesize their own folates, mammals and other eukaryotic species are devoid of THF cofactor biosynthesis and must therefore obtain them from exogenous sources. Three independent transport systems are currently known to mediate the uptake of folates and antifolates in mammalian cells:
a) The predominant cellular transport system of reduced folate cofactors is the reduced folate carrier (RFC). The RFC (also known as solute carrier family 19 member 1, SLC19A1) is a ubiquitously expressed ˜85 kDa membrane glycoprotein functioning as a bi-directional facilitative carrier that mediates the uphill transport of reduced folates by exchanging organic phosphates such as adenine nucleotides that are known to accumulate to very high intracellular levels as well as thiamine mono- and pyrophosphate. RFC displays a high-affinity for THF cofactors including leucovorin (5-formyl-THF; Kt=1 μM), while harboring only a very poor transport affinity (Kt=200-400 μM) for folic acid, an oxidized folate.
b) Another route of folate uptake is the proton-coupled folate transporter (PCFT, also known as SLC46A) which has recently been cloned. PCFT appears to be expressed independently of the RFC, functions optimally at acidic pH (5.5) and mediates the influx of both oxidized (e.g. folic acid) and THF cofactors (i.e. reduced folates) as well as various hydrophilic antifolates including MTX. PCFT, which shows an optimal transport of folates and antifolates at acidic pH (5.5) but none at physiological pH (7.4), has a key role in the absorption of both folates and antifolates in the upper small intestine.
c) The third transport route, on which the present invention is based, involves folate receptors (FRs). FRs are high-affinity folate-binding glycoproteins encoded by three distinct genes FRα (FR alpha), FRβ (FR beta) and FRγ (FR gamma). FRα □(or FR-alpha) is also known as Adult Folate Binding Protein or FDP, as Folate Receptor1 or FOLR (in mice folbp1), and as Ovarian cancer-Associated Antigen or MOv 18. FRβ (or FR beta) is also known as FOLR2 (fetal) and as FBP/PL-1(placenta). FRγ (or FR gamma) is also known as FOLR3 and as FR-G (reviewed by M. D. Salazar and M. Ratnam, Cancer Metastasis Rev. 2007 26(1), pp. 141-52.). The mature FRs, which are well-characterized, are homologous proteins with ˜70-80% amino acid identity and contain 229 to 236 amino acids as well as two to three N-glycosylation sites. FRα (FR alpha) and FRβ (FR beta) are membrane-bound, in particular glycosylphosphatidylinositol (GPI)-anchored, cell surface glycoproteins, whereas FRγ is devoid of a GPI anchor and is a secreted protein. FRα (FR alpha) and FRβ (FR beta) display a high affinity for folic acid (Kd=0.1-1 nM), 5,10-dideazatetrahydrofolic acid (DDATHF; lometrexol; Ki=0.4-1.3 nM using [3H]folic acid as a substrate) and BGC945 (which is a cyclopenta[g]quinazoline-based, thymidylate synthase inhibitor specifically transported solely via FRα (FR alpha) and not via the reduced folate carrier) (Kd=1 nM), but much lower affinity for MTX (Kd>100 nM). FR-dependent uptake of folate and antifolates proceeds via a classical mechanism of receptor-mediated endocytosis. Gene knockout studies have shown that FRα (FR alpha) (also known as Folbp1 in mice) is essential for early embryonic development and maternal folate supplementation rescued from in utero embryonic lethality and allowed for normal development.
There is an ongoing need for a safe, highly effective and cost-efficient selection system which overcomes one or more of the disadvantages of the selection systems known up to date.